Locomotion is a fundamental and vital animal behavior which relies on the activities of diverse neuronal classes residing within the brainstem and spinal cord. Spinal motor neurons play a central role in coordinating locomotor behaviors, and are engaged by networks of local interneurons that facilitate the rhythm and pattern of limb muscle activation. The basic genetic programs that govern connectivity between spinal interneurons and motor neurons are poorly understood due in large part to the complexity of the hundreds of muscle groups targeted by spinal locomotor circuits. In this exploratory proposal, we will assess the composition and connectivity of locomotor circuits in the little skate Leucoraja erinacea, a primitive cartilaginous fish that displays walking behaviors highly similar to those of tetrapods. Remarkably, we have found that the molecular profiles of fin-innervating motor neurons in Leucoraja are nearly identical to those of tetrapods. Leucoraja generates bipedal locomotion using 8 anatomically well-defined pelvic fin muscles, although the neural circuits that generate this behavior are uncharacterized. In this proposal we will determine the subtype identities and connectivity of spinal motor neurons, interneurons, and muscle in Leocoraja. We will also explore the utility of Leucoraja for assessing motor circuit connectivity programs using viral trans-synaptic tracing assays and electrophysiology. In Aim 1 we will assess the circuit composition and connectivity of motor neurons and interneurons in Leucoraja, and explore the role signaling through Hox transcription factors in the regional allocation of motor neuron columnar and pool subtype identities. In Aim2 we will use viral tracing assays to determine the pattern of connectivity between spinal interneurons and motor neurons, and explore the workings of the intraspinal central pattern generators (CPGs) that coordinate the activation of fin muscles during walking and swimming. By exploiting the relatively simple neuromusculature architecture of this primitive vertebrate, and building off our in depth knowledge of neural specification programs in the mammalian spinal cord, these studies could provide insights into the basic mechanisms through which motor circuits are assembled.